frequently asked questions

We are here to help you enjoy your scientific exploration. Please read these FAQs before contacting us. If your question still has not been answered, send us an email at support@thewellbio.com

General Questions (15)

VitroGel® is a xeno-free functional hydrogel system that closely mimics the natural extracellular matrix (ECM) environment. Modified with multiple cellular functional ligands, VitroGel allows for a robust 3D cell culture platform and can be used as an injectable delivery system for drug discovery, tissue engineering, cell therapy, and personalized medicine.

VitroGel comes in two different variations, ready-to-use, and high concentration. VitroGel Hydrogel Matrix is a ready-to-use VitroGel system preferred by many scientists for 3D and 2D cell culture. For scientists wanting full control over tunability and their cell culture environment, VitroGel High Concentration is recommended.

Ready-To-Use Hydrogel

VitroGel® Hydrogel Matrix (Cat# VHM01) – For many users, this is the preferred hydrogel for 3D cell culture and 2D coating. It is a formulation of multi-functional ligands and concentration to support a wide range of cell types for different applications. It is a ready-to-use hydrogel where you need only to add your cells, cover medium, and incubate.

High Concentration Hydrogels

For more advanced users, the VitroGel High Concentration hydrogels give scientists full control to manipulate the biophysical and biological properties of the cell culture environment. We have many types of hydrogel with different binding ligands, making our system a “Mix and Match” where you can control what is in the hydrogel for your 3D cell culture. All the high concentration hydrogels come with VitroGel Dilution Solutions to adjust the final hydrogel strength from 10 to 4000 Pa. Customized hydrogels can reach more than 20,000 Pa.

VitroGel® 3D High Concentration Kit is a pure and unmodified hydrogel that allows the maximum flexibility to manipulate the 3D cell culture environment for different needs. The unmodified hydrogel matrix structure is good for cell spheroid formation, suspension cells or cells requiring low cell-matrix interactions.

VitroGel® RGD High Concentration Kit is modified with a high concentration of RGD cell adhesive peptides, promoting the cell attachment and cell-matrix interactions during the 3D cell culture. It can achieve high levels of integrin-binding activities to promote intercellular networks even after hydrogel dilution.

VitroGel® COL High Concentration Kit is modified with collagen-mimetic peptide (GFOGER), which specifically binds the integrin α2β1, promoting many bioactivities such as osteoblastic differentiation in vitro and enhancing osteoblastic activity in vivo.

VitroGel® IKVAV High Concentration Kit is modified with laminin-derived functional peptide IKVAV, which is actively involved in different biological activities such as neuronal progenitor cell differentiation, promoting cell adhesion, neurite outgrowth, angiogenesis, and tumor growth.

VitroGel® YIGSR High Concentration Kit is modified with laminin-derived functional peptide (YIGSR), which involves endothelial cell adhesion, cell proliferation and motility/migration.

VitroGel® MMP High Concentration Kit is modified with Matrix Metalloproteinases (MMP) for a biodegradability matrix.

We can customize our hydrogels with different functional ligands. Please contact at support@thewellbio.com if you need a customized product.

The hydrogel is a xeno-free synthetic polysaccharide-based hydrogel system.

VitroGel Dilution Solution Type 1 contains sucrose to maintain the best osmolarity. Dilution Type 2 is sucrose-free. For most cell lines, VitroGel Dilution Solution Type 1 is recommended. VitroGel Dilution Solution Type 2 is for scientists working with cells that are sensitive to sugar.

Yes, all VitroGel hydrogels are tested with a dynamic rheometer for viscoelastic properties. For VitroGel High Concentration hydrogels, the elastic modulus (G’) can be adjusted from 10 to 4,000 Pa by changing the hydrogel concentration with the VitroGel Dilution Solution.

VitroGel does not contain any natural ECM proteins. Scientists can add growth factors and proteins to the hydrogel system for a well-defined system.

We test the elastic modulus (G’) of VitroGel with a dynamic rheometer. The G’ is dependent on the hydrogel dilution and the cell culture medium used. The G’ of non-diluted VitroGel High Concentration is around 4,000 Pa. If a higher stiffness is required, please contact us at support@thewellbio.com for a customized hydrogel that can reach over 20k Pa.

Adjusting the temperature will not induce the hydrogel formation. VitroGel can maintain the liquid form at room temperature. The hydrogel formation can be induced by mixing with an ionic solution such as a cell culture medium. Increasing the temperature would reduce the viscosity of the hydrogel solution.

Yes. VitroGel supports the diffusion of molecules of different sizes from small molecules to big proteins (e.g., IgG). Drug compounds, antibodies, staining dyes can be added on the cover medium to penetrate the hydrogel. VitroGel can be mix with molecules for control release experiments.

VitroGel is a xeno-free system. We do not detect any autofluorescence in our various imaging testing. We recommend scientists perform a control experiment to determine background fluorescence.

Yes, scientists can easily recover cells with our enzyme-free, ready-to-use VitroGel Cell Recovery Solution. VitroGel Cell Recovery Solution is room temperature stable with a neutral pH and works at 37°C operating temperature. The recovery solution can maintain high cell viability during the recovery process. Harvested cells can be sub-cultured in both 2D and 3D cultures within 20 minutes.

Additional information can be found here.

Yes, VitroGel can support 3D cell culture, 2D thick gel coating and 2D thin gel coating culture.  Embedding cells in the hydrogel matrix (3D cell culture) can maximize the cell-matrix interaction and potential cell-cell communication in response to different biophysical and biological properties of the hydrogel. Some cells are hard to culture in 3D; as a result, 2D coating is an alternative way.

2D thick gel coating uses a layer of hydrogel to change the properties of the substance. It is a method between the traditional 2D culture and the true 3D cell culture. 2D thick gel coating is an alternative approach if scientists do not want to jump into 3D cell culture but want to see how cells behave differently based on different substances. It is also a good method for layer by layer co-culture as well.
2D thin gel coating is used as a thin layer of hydrogel to change the surface properties of the substance, which may change the capability for cell attachment but not the stiffness of the substance.

Yes, VitroGel is biocompatible and safe for animal study.

We have tested the growth of cells in the hydrogel system for more than eight weeks. Depending on the cell type and application, the 3D cell culture can last even longer. Scientists may need to change the cover media more frequently once the number and size of cells increases.

No specific plate is required. Any regular tissue culture treated plate can be used.

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Hydrogel Formation and Preparation Protocols (11)

The hydrogel formation starts when VitroGel is mixed with the cell culture medium.  The hydrogel molecules will interact with the ionic molecules, such as Ca2+ or Na+, from the cell culture medium to induce a matrix structure (hydrogel). For 3D culture operation, the hydrogel formation is in two stages: a) soft hydrogel formation, b) hydrogel stabilization.

  1. Soft hydrogel formation: The hydrogel formation process is slow when a small amount of cell culture medium is used. At this stage, the hydrogel is soft and possess a shear-thinning and rapid recovering mechanical property, which makes the hydrogel injectable for in vivo The slow hydrogel-forming process and the injectable property of the soft hydrogel create a time frame for easy hydrogel transfer from the mixing tube to the cell culture plate.
  2. Hydrogel stabilization: After soft hydrogel formation, adding additional cell culture medium on top of the hydrogel would allow more ionic molecules to penetrate the hydrogel matrix and further saturate the hydrogel cross-linking. A solid hydrogel would form during this process.

More information can be found here.

Adjusting the mixing ratio between the VitroGel solution and cell culture medium would change the hydrogel formation rate.

If the hydrogel solidifies too fast after mixing with the culture medium (showing as small solid gel chunk), adjust the mixing ratio by using less cell culture medium. For example, if mixing 2 mL hydrogel solution with 0.5 mL cell culture medium leads to solid gel chuck (particles), then mixing 2 mL diluted hydrogel solution with 0.2-0.4 mL cell culture medium would help to solve the issue.

On the other hand, if the hydrogel formation is too slow, adjust the mixing ratio by using more cell culture medium. For example, if mixing 2 mL diluted hydrogel solution with 0.5 mL cell culture medium leads to a slow hydrogel formation, then mixing 2 mL diluted hydrogel solution with 1-2 mL cell culture medium would help to solve the issue.

The bubble issue is related to the increased solution viscosity after mixing the gel solution with cell medium. Here are some suggestions that can help to reduce the formation of bubbles:

  1. Warm up the VitroGel solution to 37° C to reduce the viscosity of the gel.
  2. Gently mix the VitroGel solution with the cell medium. Then, pipette slowly without introducing bubbles.
  3. Quickly spin the mixing tube to get rid of bubbles.

Yes. Extracellular matrix proteins or other molecular compounds can be added into the VitroGel system. Before hydrogel formation, add the proteins or the molecular compounds into the cell culture medium and then mix directly with the VitroGel 3D hydrogel solution. Please note that the hydrogel formation time and the final gel stiffness might change due to the salts contained in the proteins or chemical compounds. Please contact us at support@thewellbio.com if you have any questions or concerns about adding additional compounds to the VitroGel system.

This issue might be because of the following reasons:

  1. Using a non-treated tissue culture plate, which has a more hydrophobic surface. This reduces the attachment of hydrogel/cells on the surface of the well-plate. For better performance, we suggest using a treated tissue culture plate with VitroGel.
  2. Adding the hydrogel as a dome instead of covering the whole bottom of the well plate might also cause this issue. We suggest gently tilting and swirling the well plate after adding the hydrogel to ensure the whole bottom of the well plate is covered by the gel.
  3. Not waiting long enough before adding the additional medium on the top of the hydrogel. After transferring the hydrogel to the well plate, please wait 10-30 minutes for hydrogel stabilization before adding the top medium. Adding the medium before the hydrogel stabilizes would disrupt the structure of hydrogel. The lower the concentration of the hydrogel, the longer the waiting time is needed.
  4. After the initial soft hydrogel formation, it is crucial to make sure the hydrogel is stable and attached to the bottom of the well plate before adding the cover medium. If the hydrogel is not stable, it might detach from the bottom of the well after adding the cover medium. During the hydrogel formation, the gel is soft; do not shake the plate or position the plate vertically. Keep the plate horizontally.

Adding the hydrogel as a dome shape is not recommended. The dome shape may not stick to the bottom of the culture plate for long term culture. The hydrogel should cover the whole bottom of the well plate. We suggest gently tilting/swirling the well plate after adding the hydrogel to ensure the gel coats the entire bottom of the well plate. Using a 96 well plate can reduce the usage volume of the hydrogel to 30-75 µL/well. There is no issue with molecular penetration when adding the cover medium to feed the cells.

Yes, serum-free medium works with VitroGel.

For the High Concentration hydrogels, it is not recommended.  The concentration of the ionic molecule in medium would be much higher than the dilution solution, so diluting VitroGel with cell culture medium may cause the gel to form too quickly and chunky. We recommend using VitroGel Dilution Solution to adjust the highly concentrated VitroGel.

Alternatively, VitroGel Hydrogel Matrix (Cat# VHM01) is a better option for scientists who do not want to worry about dilution and only want to mix the hydrogel with cell suspension directly.

After mixing with the cell culture medium, we recommend transferring the mixture to the tissue culture plate within 10 minutes. The hydrogel formation starts after mixing the VitroGel solution with the cell culture medium.

If you have multiple samples with different hydrogel conditions or cell types to prepare, we recommend transferring the mixture of sample 1 to the tissue culture plate before mixing the hydrogel with cell culture medium for sample 2.

Yes, it is fine to place the gel mixture in an incubator instead of room temperature, but please note, the gel forms a little slower in an incubator than at room temperature. As a result, for lower concentrated gels, you will need to keep it in an incubator for a little longer. However, for gels at a higher concentration or VitroGel Hydrogel Matrix, it is not a problem.

Although, for most cases, pre-coating the culture plate is not necessary, coating the plate with 1X PBS or 10-100 mM CaCl₂ can improve the hydrogel attachment. Please check the following protocols for coating the culture plate:

  1. Add the PBS or CaCl₂ solution to the culture plate for 30 min.
  2. Remove the PBS or CaCl₂ solution, open the lid under the biosafety hood for 10-20 min before adding the hydrogel. The recommend volumes of PBS or CaCl₂ for different sizes of well plates are list in Table 1 below.
    Table 1: Recommend volume of PBS or CaCl₂ solution for pre-coating well plate

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Cell Preparation and Culture on VitroGel (9)

Cells can be prepared in regular complete cell culture medium and be directly mixed with VitroGel. If you want to adjust the concentration of the serum or other critical supplement in the final hydrogel, follow the steps below:

If cells cultured in complete cell culture medium, which is supplement with 10% FBS or other critical supplements, please prepare the cell suspension using the following methods before mixing it with hydrogel solution.

  1. Prepare the cell suspension with 2X concentration (e.g. 100K) and mix with 100% FBS at 1:1 (v/v) ratio to get 1X cell suspension (e.g. 50K) with 50% FBS.
  2. Mix VitroGel hydrogel solution with the cell suspension from above at 4:1 (v/v) ratio to get the final cells in the hydrogel at 10K with 10% FBS supplement.

Note: Do not make 2X (or higher) concentration medium. The ionic molecules in the medium would affect hydrogel formation. The high concentration medium might make gel chunky or non-uniform.

The serum or critical supplement in the hydrogel would affect cell growth, especially during the first 48 hrs. Please see the example figures below.

Bone marrow cells (OP9) 3D cultured in VitroGel LDP3 with 2% and 10% FBS. The cells were encapsulated in hydrogel matrix with 2% and 10% final FBS concentrations respectively. The images were taken 18 hours after cell seeding

The final cell concentration can be optimized based on different cell types. We recommend preparing cell suspension at the following concentration for:

  • 3D cell culture:5-2 x 106 cells/mL (the cell suspension need to mix with VitroGel solution at the 4:1 ratio (VitroGel solution: Cell suspension at 4:1 v/v), which make the final cell concentration in the hydrogel is 1-4 x 105 cells/mL)
  • 2D hydrogel coating: 1-5 x 105 cells/mL

Typically, we recommend changing the cover medium every other day, similar to regular 2D cell culture. However, it depends on the experiment’s needs. Some experiments might require changing every 24 hours and some might not require changing for an entire week. Please contact us at support@thewellbio.com if you are unsure of this.

Changing 100% of the cover medium might cause the disruption of the hydrogel. We recommend adding additional fresh medium without removing the top medium for the first medium change. Afterward, change 50-80% of the cover medium. (Please check the recommended volume of additional cover medium in Table below.)

Yes, the medium on top can penetrate through the hydrogel.

Yes, the mobility of cells on VitroGel can be observed in both 3D culture and 2D hydrogel coating culture. Spheroid invasion assay, wound healing assay, or 3D cell migration can be performed with VitroGel.

Yes. Cells can be harvested from the VitroGel system by using the VitroGel Cell Recovery Solution and be sub-cultured for an additional period by using fresh VitroGel.

The co-culture of two or more different cell types can be performed with the VitroGel system. Besides adding different cell types together with the hydrogel solution for co-culture, each cell type can also be mixed with the hydrogel solution and be added layer by layer. After the first layer of hydrogel become stable, carefully overlay the second layer of cells/hydrogel mixture on top of the first layer of cells/hydrogel.

Normally, spheroid formation requires about 3 – 10 days after culture in the hydrogel system. The formation time may vary depending on different cell types. The small cell aggregation (colony formation) might happen overnight.

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Imaging and Analysis (4)

Yes. Cells can be stained within the hydrogel or harvested from the hydrogel and then stain. Most fluorescent dyes and immunological reagents can be used at standard protocols. VitroGel hydrogel is transparent and compatible with different imaging systems for cell observation.

Yes. The cells can be fixed inside the hydrogel with PFA. Immunolabeling of cells works well with VitroGel.

Yes, VitroGel is compatible with many methods to run the live/dead and cell proliferation assay. The live/dead cell staining kit can be used for image analysis. The MTT, MTS, ATP or DNA assay can be used for cell proliferation. You can also harvest cells out of the hydrogel by using our VitroGel Cell Recovery solution and run the trypan blue or AO/PI assay.

Yes. Cells can be harvested from the hydrogel and be used for molecular analyses according to standard procedures. The hydrogel is transparent, and you can use the molecular assay directly with the hydrogel. Additionally, the hydrogel can be easily dissolved by using a homogenizer/ultrasonic processor, so scientists can lysis cells together with hydrogel to extract DNA/RNA.

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Applications (8)

Yes, the VitroGel hydrogels are a great system to build 3D cell models for drug/compound screening. VitroGel is compatible for high-throughput screening with an auto liquid handling machine. Molecules can be mixed with the hydrogel or be added directly from the top of the hydrogel. This will allow an easy diffusion through the gel.

Yes, VitroGel can be used to perform 3D transfection studies.

Yes, VitroGel can be added directly on top of the cells or tissue slide.

Yes, VitroGel can be injected before or after soft hydrogel formation for in vivo study.
Before the hydrogel formation:   The VitroGel solution can be injected directly into the animal. It will become a hydrogel when it encounters the ionic compounds of the physiological environment.

After soft hydrogel formation:  The VitroGel hydrogel has an advanced injectable property after soft hydrogel formation. Mixing the hydrogel solution and cell culture media/PBS at a proper ratio, the final hydrogel becomes injectable for in vivo studies. Using this method, cells or other chemical compounds can be mixed in the hydrogel before injection.

Yes, VitroGel is an excellent delivery system for cell therapy. Using VitroGel as an injectable delivery system, scientists can achieve better cell retention and higher cell viability for cell therapy.

We normally use 18-30 G needle size.

Yes, DMSO can be used with VitroGel.  For the maximum amount of DMSO, we suggest no more than 50% of the volume.

Yes, VitroGel can be used for cryopreservation.  It can help to promote cell viability.

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